Verotoxin pharmaceutical compositions and medical treatments therewith

ABSTRACT

Pharmaceutical compositions comprising known verotoxins, particularly, verotoxin 1, have been found to be useful in the treatment of mammalian neoplasia, particularly, ovarian cancer and skin cancer. Surprisingly, although verotoxin 1 has previously been shown to have anti-neoplastic activity in vitro, non-lethal doses of verotoxin 1 have been shown to be therapeutically anti-neoplastic in vivo.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 08/386,957, filedFeb. 10, 1995 (now abandoned).

FIELD OF THE INVENTION

This invention relates to verotoxin pharmaceutical compositions and tomethods of treating mammalian neoplasia, particularly, ovarian and skincancers, therewith.

BACKGROUND TO THE INVENTION

Bacteriocins are bacterial proteins produced to prevent the growth ofcompeting microorganisms in a particular biological niche. A preparationof bacteriocin from a particular strain of E. coli (HSC₁₀) has long beenshown to have anti-neoplastic activity against a variety of human tumourcell lines in vitro (1,2). This preparation, previously referred to asPPB (partially purified bacteriocin (2)) or ACP (anti-cancer proteins(2)) was also effective in a murine tumour model, of preventingmetastases to the lung (2).

Verotoxins, also known as SHIGA-like toxins, comprise a family known asVerotoxin 1, Verotoxin 2, Verotoxin 2c and Verotoxin 2e of subunittoxins elaborated by some strains of E. coli (3). These toxins areinvolved in the etiology of the hemolytic uremic syndrome (3,4) andhaemorrhagic colitis (5). Cell cytotoxicity is mediated via the bindingof the B subunit of the holotoxin to the receptor glycolipid,globotriaosylceramide, in sensitive cells (6).

The verotoxin family of E coli elaborated toxins bind to the globoseries glycolipid globotriaosylceramide and require terminal gal α-1-4gal residue for binding. In addition, VT2e, the pig edema disease toxin,recognizes globotetraosylceramide (Gb₄) containing an additional β 1-3linked galNac residue. These glycolipids are the functional receptorsfor these toxins since incorporation of the glycolipid into receptornegative cells renders the recipient cells sensitive to cytotoxicity.The toxins inhibit protein synthesis via the A subunit—an N-glycanasewhich removes a specific adenine base in the 28S RNA of the 60S RNAribosomal subunit. However, the specific cytotoxicity and specificactivity is a function of the B subunit. In an in vitro translationsystem, the verotoxin A subunit is the most potent inhibitor of proteinsynthesis yet described, being effective at a concentration of about 8pM. In the rabbit model of verocytotoxemia, pathology and toxintargeting is restricted to tissues which contain the glycolipid receptorand these comprise endothelial cells of a subset of the bloodvasculature. Verotoxins have been strongly implicated as the etiologicalagents for hemolytic uremic syndrome and haemorrhagic colitis,microangiopathies of the glomerular or gastrointestinal capillariesrespectively. Human umbilical vein endothelial cells (HUVEC) aresensitive to verotoxin but this sensitivity is variable according tocell line. Human adult renal endothelial cells are exquisitely sensitiveto verotoxin in vitro and express a correspondingly high level of Gb₃.However, HUS is primarily a disease of children under three and theelderly, following gastrointestinal VTEC infection. It has been shownthat receptors for verotoxin are present in the glomeruli of infantsunder this age but are not expressed in the glomeruli of normal adults.HUVEC can be sensitized to the effect of verotoxin by pretreatment bytumour necrosis factor which results in a specific elevation of Gb₃synthesis (7,8). Human renal endothelial cells on the other hand,although they express high levels of Gb₃ in culture, cannot bestimulated to increase Gb₃ synthesis (8). It has been suggested that thetransition from renal tissue to primary endothelial cell culture invitro results in the maximum stimulation of Gb₃ synthesis from a zerobackground (9). We therefore suspect that HUS in the elderly is theresult of verotoxemia and a concomitant stimulation of renal endothelialcell Gb₃ synthesis by some other factor, e.g. LPS stimulation of serum αTNF. Thus under these conditions, the majority of individuals (exceptingthe very young) would not be liable to VT induced renal pathologyfollowing systemic verotoxemia.

It has also shown that the verotoxin targets a sub-population of human Bcells in vitro (10). These Gb₃ containing B cells are found within thegerminal centres of lymph nodes (11). It has been proposed that Gb₃ maybe involved in a germinal centre homing by CD19 positive B cells (12)and that Gb₃ may be involved in the mechanisms of antigen presentation(13).

Elevated levels of Gb₃ have been associated with several other humantumours (14-16), but ovarian tumours have not been previouslyinvestigated. Gb₃ is the p^(k) blood group antigen (17). Tissue surveysusing anti-p^(k) antisera have shown that human ovaries do not expressthis glycolipid (18, 19).

Sensitivity to VT1 cytotoxicity in vitro has been shown to be a functionof cell growth, the stationary phase cells being refractile tocytotoxicity (20). The sequence homology between the receptor binding Bsubunit and the human α2-interferon receptor and the B cell marker CD19suggests that expression of Gb₃ is involved in the mechanism ofα2-interferon and CD19 signal transduction (12). On surface ligation,Gb₃ has been shown to undergo a retrograde intracellular transport viathe rough endoplasmic reticulum to the nuclear membrane (21).

REFERENCE LIST

The present specification refers to the following publications, each ofwhich is incorporated herein by reference:

1. Farkas-Himsley, H. and R. Cheung. Bacterial Proteinaceous Products(bacteriocins as cytotoxic agents of neoplasia). Cancer Res.36:3561-3567, (1976).

2. Hill, R. P. and H. Farkas-Himsley. Further studies of the action of apartially purified bacteriocin against a murine fibrosarcoma. CancerRes. 51:1359-1365 (1991).

3. Karmali, M. A. Infection by Verocytotoxin-producing Escherichia coli.Clin. Microbiol. Rev. 2:15-38 (1989).

4. Karmali, M. A., M. Petric, C. Lim, P. C. Fleming, G. S. Arbus and H.Lior, 1985. The association between hemolytic uremic syndrome andinfection by Verotoxin-producing Escherichia coli, J. Infect. Dis.151:775.

5. Riley, L. W., R. S. Remis, S. D. Helgerson, H. B. McGee, J. G. Wells,B. R. Davis, R. J. Hebert, E. S. Olcott, L. M. Johnson, N. T. Hargrett,P. A. Blake and M. C. Cohen. Haemorrhagic colitis associated with a rareEscherichia coli serotype. N. Engl. J. Med. 308:681 (1983).

6. Lingwood, C. A., Advances in Lipid Research. R. Bell, Y. A. Hannunand A. M. Jr. Academic Press. 25:189-211 (1993).

7. van de Kar, N. C. A. J., L. A. H. Monnens, M. Karmali and V. W. M.van Hinsbergh. Tumour necrosis factor and interleukin-1 induceexpression of the verotoxin receptor globotriaosyl ceramide on humanendothelial cells. Implications for the pathogenesis of the HemolyticUremic Syndrome. Blood. 80:2755, (1992).

8. Obrig T., C. Louise, C. Lingwood, B. Boyd, L. Barley-Maloney and T.Daniel. Endothelial heterogeneity in Shiga toxin receptors andresponses. J. Biol. Chem. 268:15484-15488 (1993).

9. Lingwood, C. A. Verotoxin-binding in human renal sections. Nephron.66:21-28 (1994).

10. Cohen, A., V. Madrid-Marina, Z. Estrov, M. Freedman, C. A. Lingwoodand H. M. Dosch. Expression of glycolipid receptors to Shiga-like toxinon human B lymphocytes: a mechanism for the failure of long-livedantibody response to dysenteric disease. Int. Immunol. 2:1-8 (1990).

11. Gregory, C. D., T. Turz, C. F. Edwards, C. Tetaud, M. Talbot, B.Caillou, A. B. Rickenson and M. Lipinski. 1987. Identification of asubset of normal B cells with a Burkitt's lymphoma (BL)-like phenotype.J. Immunol. 139:313-318 (1987).

12. Maloney, M. D. and C. A. Lingwood, CD19 has a potential CD77(globotriaosyl ceramide) binding site with sequence similarity toverotoxin B-subunits: Implications of molecular mimicry for B celladhesion and enterohemorrhagic E. coli pathogenesis. J. Exp. Med. 180:191-201, (1994).

13. Maloney, M. and C. Lingwood. Interaction of verotoxins withglycosphingolipids. TIGG. 5:23-31 (1993).

14. Li, S. C., S. K. Kundu, R. Degasperi and Y. T. Li. Accumulation ofglobotriaosylceramide in a case of leiomyosarcoma. Biochem. J.240:925-927 (1986).

15. Mannori G., O. Cecconi, G. Mugnai and S. Ruggieri. Role ofglycolipids in the metastatic process: Characteristics neutralglycolipids in clones with different metastatic potentials isolated froma murine fibrosarcoma cell line. Int. J. Cancer. 45:984-988 (1990).

16. Ohyama, C., Y. Fukushi, M. Satoh, S. Saitoh, S. Orikasa, E.Nudelman, M. Straud and S. I. Hakomori. Changes in glycolipid expressionin human testicular tumours. Int. J. Cancer. 45:1040-1044, (1990).

17. Naiki, M. and D. M. Marcus. Human erythrocyte P and P^(k) bloodgroup antigens: Identification as glycosphingolipids. Biochem. Biophys.Res. Comm. 60:1105-1111, (1974).

18. Pallesen, G. and J. Zeuthen. Distribution of theBurkitt's-lymphoma-associated antigen (BLA) in normal human tissue andmalignant lymphoma as defined by immunohistological staining withmonoclonal antibody 38:13. J. Cancer Res. Clin. Oncol. 113:78-86 (1987).

19. Kasai, K., J. Galton, P. Terasaki, A. Wakisaka, M. Kawahara, T. Rootand S. I. Hakomori. Tissue distribution of the Pk antigen as determinedby a monoclonal antibody. J. Immunogenet. 12:213 (1985).

20. Pudymaitis, A. and C. A. Lingwood. Susceptibility to verotoxin as afunction of the cell cycle. J. Cell Physiol. 150:632-639 (1992).

21. Sandvig, K., O. Garred, K. Prydz, J. Kozlov, S. Hansen and B. vanDeurs. Retrograde transport of endocytosed Shiga toxin to theendoplasmic reticulum. Nature. 358:510-512 (1992).

Although anti-neoplastic effects of bacterial preparations have beenknown for over twenty years, the neoplastic effect of verotoxin per sehas, to-date, remained unknown. As a result of extensive investigations,we have discovered that verotoxin, particularly Verotoxin 1, is anactive component within the ACP and that purified Verotoxin 1 has potentanti-neoplasia effect in vitro and in vivo. Most surprisingly, we havefound effective in vivo anti-cancer treatments of human beingscommensurate with non-toxic administered dosages.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a pharmaceuticalcomposition for the treatment of mammalian neoplasia and, particularly,skin cancer and ovarian cancer.

It is a further object of the present invention to provide a method oftreating mammalian neoplasia, particularly, skin, brain and ovariancancers.

Accordingly, in one aspect the invention provides a pharmaceuticalcomposition for the treatment of mammalian neoplasia comprising anon-lethal anti-neoplasia effective amount of a verotoxin, preferably,verotoxin 1, and a suitable pharmaceutically acceptable diluent,adjuvant or carrier therefor.

The invention preferably provides a pharmaceutical composition andmethod of treatment for mammalian skin cancers, brain cancers andovarian cancer.

In a further aspect the invention provides a process for the manufactureof a pharmaceutical composition for the treatment of mammalianneoplasia, said process comprising admixing verotoxin with apharmaceutically acceptable carrier, adjuvant or diluent therefor.

The present invention provides selective, specific cancer treatmentswherein verotoxin selectively binds with Gb₃ in Gb₃-containing cells.This is in contrast to the use of broad spectrum anti-neoplastic agentssuch as most chemotherapeutic agents, in that non-Gb₃ containing cellsare not affected by verotoxin. The present invention thus provides amost beneficial, cell-selective, therapeutic treatment.

The treatment is of value against cutaneous T-cell lymphomas,particularly, Mycosis Fungoides, sezary syndrome and related cutaneousdisease lymphomatoid papilosis. For example, Mycosis fungoides lesionsin humans have been cleared without any observed adverse systemiceffects by the application of VT1 (5 ng in 2 ml. solution) byinterdermal injection in patients.

In a further aspect, the invention provides a method of treatingmammalian neoplasia comprising treating said mammal with a non-lethalanti-neoplasia effective amount of a verotoxin, preferably Verotoxin 1.

The verotoxin may be administered to the patient by methods well-knownin the art, namely, intravenously, intra-arterially, topically,subcutaneously, by ingestion, intra-muscular injection, inhalation, andthe like, as is appropriately suitable to the disease. For treatment ofa skin cancer, sub-cutaneous application is preferred.

In the practice of the present invention, Verotoxin 1 has been injectedintramuscularly into a patient with advanced ovarian carcinoma. Noadverse affects were monitored on lymphocyte or renal function and aserum tumour marker was found to continue to rise when the patient wastreated with relatively high doses of Verotoxin 1. This tumour wasrefractory to all conventional cancer therapies. No effect was found onhemoglobin levels.

The verotoxin is, typically, administered in a suitable vehicle in whichthe active verotoxin ingredient is either dissolved or suspended in aliquid, such as serum to permit the verotoxin to be delivered forexample, in one aspect from the bloodstream or in an alternative aspectsub-cutaneously to the neoplastic cells. Alternative, for example,solutions are, typically, alcohol solutions, dimethyl sulfoxidesolutions, or aqueous solutions containing, for example, polyethyleneglycol containing, for example, polyethylene glycol 400, Cremophor-EL orCyclodextrin. Such vehicles are well-known in the art, and useful forthe purpose of delivering a pharmaceutical to the site of action.

Several multi-drug resistant cell lines were found to be hypersensitiveto Verotoxin 1. For example, multidrug resistant ovarian cancer celllines SKVLB and SKOVLC were more sensitive to VT cytotoxicity thancorresponding non-multidrug resistant ovarian cancer cell line SKOV3.Such an observation indicates the possible beneficial effect forpatients bearing the SKVLB cell line cancer than those with the SKOV3cell line under VT treatment. Further, our observed binding of VT1 tothe lumen of blood vessels which vascularize the tumour mass, inaddition to the tumour cells per se, may result in an anti-angiogeniceffect to augment the direct anti-neoplastic effect of verotoxin.

BRIEF DESCRIPTION OF THE DRAWINGS

In order that the invention may be better understood preferredembodiments will now be described, by way of example only, withreference to the accompanying drawings wherein:

FIG. 1 shows the selective neutralization of ACP cytotoxicity by antiVT1 and or anti VT1 B subunit but not by anti VT2 antibodies asdetermined by cell density measurement after 48 hours;

FIG. 2 shows the viability of selected ovarian and breast tumour celllines to verotoxin concentration;

FIG. 3 represents VT1 contained within ACP preparation binding to Gb₃(and Gb₂).

FIG. 4 represents VT thin layer chromatography overlay of ovarian tumourand ovary glycolipids;

FIG. 5 represents VT thin layer chromatography overlay of selected cellline glycolipids;

FIG. 6 represents in three graphs ovarian cell line sensitivity to VT1,VT2 and VT2c;

FIG. 7 represents glioblastoma multiforme cell line sensitivity to VT1,VT2 and VT2c;

FIG. 8 represents the distribution of labelled VT1 B subunit (VTB-¹³¹I)administered IP (inter-peridinually) in a Gb₃ tumour bearing nude mouse;and

FIG. 9 represents the results of a three-day treatment of several humanastrocytoma cell lines with VT1.

DETAILED DESCRIPTION OF THE INVENTION

Experimental

The isolation and purification of verotoxins VT1, VT2 and VT2c have beenearlier described.

Verotoxin 1 was prepared genetically from the high expressionrecombinant E. coli pJB28, J. Bacteriol 166:375 and 169:4313. Thegenerally protein purification procedure described in FEMS Microbiol.Lett. 41:63, was followed.

Verotoxin 2 was obtained from R82, Infect. Immun. 56:1926-1933; (1988);and purified according to FEMS Microbiol. Lett. 48:379-383 (1987).

Verotoxin 2c was obtained from a clinical strain E32511 and purifiedaccording to FEMS Microbiol. Lett. 51:211-216 (1988).

Purification of VT1 from JB28

Pellet Preparation may be conducted as follows:

1. Prepare 6×1 L LB broth in 3×5 L jugs (media) and autoclave.

 Add carbenicillin to give a 100 μg/ml final conc. when cool.

2. Seed at least 6 ml of penassay (tubes in cold room) +100 μg/mlcarbenicillin with JB28 and incubate O/N @37° C.

3. Seed jugs (1 ml seed/liter broth) next morning and incubate for 24hours at 37° C. at 200 rpm (vigorous shaking).

4. Spin down bugs at 9K for 15 min. at 4° C. and scrape pellet into afreezer bag for future use. Freeze at −70° C.

Preparation of Crude Toxin Extract:

1. Retrieve pellet and dump into beaker. Resuspend in 400 ml of PBScontaining 0.1 mg/ml polymyxin B, 50 mg PMSF using a blender. Blendthoroughly then sonicate on ice for −1 minute to disperse further.

2. Incubate in shaking incubator, 200 rpm, or with vigorous stirring@37° C. for 1 hour.

3. Spin down cells @9K for 15 minutes.

4. Pour off supernatant and keep. Resuspend pellet in 400 ml PBS with0.1 mg/ml polymyxin B and PMSF. Blend and sonicate as before.

5. Incubate with vigorous shaking/stirring at 37° C. for 1 hour.

6. Spin at 10K for 15 minutes and save supernatant.

7. The supernatants should be quite yellow and the bacterial pelletshould become more fine and diffuse with each extraction step.

8. Filter the combined supernatants through Whatman filter paper thanthrough a glass fibre filter to clarify. This step is optional, but willgreatly speed the concentration step.

9. Amicon the combined supernatants at 70 psi (max.) using a YM10membrane (takes about 200 hours) to concentrate to <50 ml.

Chromatography:

Hydroxylapatite

1. Equilibrate hydroxylapatite column with 10 mM K or Na phosphate(several column volumes).

2. Load sample and wash with equilibration buffer until absorbance ofeffluent is negligible.

3. Add 2 column volumes (150 ml) of 100 mM K phosphate (untilyellow-coloured fractions emerge) and collect 3 ml fractions.

4. Wash column with 500 mM K phosphate and re-equilibrate with 10 mM Kphosphate. Add 0.05% sodium azide.

Chromatofocussing

5. Measure fractions (A₂₈₀) and Pool peak fractions from HA.

6. Dialyse against 2 L 0.025M imidazole-HCI pH 7.4 O/N. Also equilibratethe chromatofocussing column O/N with the same (300 ml).

7. Load sample and follow with 400 ml polybuffer-HCI pH 5.0 (50 mlpolybuffer 74+350 ml dH₂O, a 1:7 dilution, −pH to 5.0 with HCI). NOTE:make sure the sample is equilibrated to the temperature that the columnwill be run at (usually room temperature) prior to loading. If thecolumn is to be run at 4° then buffers must be pH'd at 4° C. and thecolumn equilibrated at this temperature.

8. Collect 1 ml fractions and test them for A₂₈₀ and pH.

9. Plot the A₂₈₀ and pool peak fractions at about pH 6.8 for VT-1 (poolside peaks separately).

10. Clean column with 100 ml 1M NaCl. if really dirty follow with 100 ml1M HCI, but quickly equilibrate column with imidazole. Store column in20% ethanol in 25 mM imidazole.

Cibachron blue

11. Equilibrate cibachron blue with 10 mM Na phosphate buffer, pH 7.2(100 ml).

12. Load sample directly from CF and follow with 60 ml of same buffer.

13. Elute with 0.5M NaCl in above buffer and collect fractions.

14. Test fractions for A₂₈₀ and cytotoxicity and pool appropriate ones.

15. Clean column with 25 ml each of 8M Urea in wash buffer and 1M NaCIin wash buffer.

16. Reequilibrate column with 10 mm phosphate containing 0.1% azide.

17. Dialyse peak fractions against wash buffer with one change.

18. Lyophilize and resuspend in 1 ml dH₂O.

19. Do protein assay and run SDS-PAGE to check purity.

Solutions:

HA column potassium phosphate buffer (0.5M stock) 17.42 g K₂HPO₄ up to300 ml with dH2O 6.8 g KH₂PO₄ pH 7.2 with KOH CF column imidazole buffer0.851 g/500 ml H₂O pH 7.4 with HCI CB column sodium phosphate buffer(Wash buffer-WB) 0.71 g/500 ml Na₂HPO₄ pH 7.2 with HAc degas Elutionbuffer Cleaning Buffers 2.922 g NaCI/100 ml WB 12.012 g Urea/25 ml WB1.461 g NaCI/25 ml WB

Purification of VT2 from R82

Pellet Preparation:

1. Prepare 3×2 L penassay broth (Antibiotic Meida 3, DIFCO; pH 7.0) in3×5 L jugs and autoclave at 121° C. for 20 minutes. Allow broth to coolto room temperature before use.

2. Seed minimum 3×2 ml of penassay broth containing 75 μg/mlcarbenicillin (Disodium salt, SIGMA) with R82 and incubate overnight at37° C., with shaking.

3. Add 50 μg/ml carbenicillin to each of the 5 L jugs (from step 1).Seed each jug with 2 ml of seed (step 2) and incubate for 24 hours at37° C. with shaking of approximately 120 rpm.

4. Heat incubator to 45° C. and incubate for 30 minutes.

5. Reduce temperature to 37° C. and incubate for another 3 hrs.

6. Spin down culture solution at 9,000× g for 15-20 min at 4° C. Discardsupernatant and store pellets at −20° C.

Preparation of Crude Toxin Extract:

1. Resuspend pellets in 100 ml of PBS (phosphate buffered saline, OXOID;pH 7.3).

2. Add 0.3 mg/ml PMSF (phenylmethyl-sulfonyl fluoride, SIGMA) dissolvedin 0.5 ml acetone to pellet solution. Let acetone evaporate. Sonicate onice at highest output possible for 5 min or until an homogeneoussolution is obtained.

3. Spin down cell at 9,000× g at 4° C. for 20 min. Discard pellets.

4. Concentrate supernatants using ultrafiltration (Model 8400 standardinfiltration cell, AMICON) with N₂ no higher than 70 psi and using a10,000 MW cutoff membrane filter (YM10 membrane, AMICON).

5. Using 12-14,000 MW cutoff tubing (SPECTRAPOR) (now and in alldialysis steps), dialyse toxin solution against 4 L of 10 mM potassiumphosphate overnight, with stirring at 4° C.

Chromatography:

Hydroxylapatite (HA)

1. Equilibrate hydroxylapatite column (BSA binding capacity: 32 mg/g,approximately 113 ml volume; CALBIOCHEM (BEHRING DIAGNOSTICS)) with 2column volumes of 10 mM potassium phosphate.

2. Load sample and follow with 1 column volume 10 mM potassiumphosphate.

3. Add 2 column volumes of 200 mM potassium phosphate and collect 2 mlfractions. The fractions containing the toxin should be coloureddifferently from the other fractions.

4. Wash column with 1 column volume of 500 mM potassium phosphate andreequilibrate with 1 column volume of 10 mM potassium phosphate. Addazide to the top of the column for storage.

Chromatofocussing (CF)

5. Pool peak fractions from HA column either by colour or bycytotoxicity test on Vero cells (10-fold dilutions).

6. Dialyse pooled fractions against 4 L 0.025M Histidine-HCl pH 6.2(SIGMA) overnight. Also equilibrate the chromatofocussing column (PBE(polybuffer exchanger) 94, 1.5 cm diameter, 57 ml volume; PHARMACIA)overnight with the same buffer (300 ml).

7. Loan sample and follow with 400 ml polybuffer-HCl pH 4.0 (50 mlpolybuffer 74 (PHARMACIA)+350 ml dH₂O−pH to 4.0 with HCl).

8. Collect 2 ml fractions and test the pH of each fraction. Once the pHhas dropped to 3.95, stop collecting fractions. Test the fractions usingabsorbance of 280 nm or by cytotoxicity on Vero cells (10-folddilutions).

9. Pool peak fractions, and return pH to 7.0 using 1N NaOH.

10. Clean column with 200 ml 1M NaCl. If dirty follow with 100 ml 1MHCl, but quickly equilibrate column with 0.025M imidazole, otherwiseequilibrate with 24% EtOH-H₂O.

Cibachron blue (CB)

11. Equilibrate cibachron blue (2 cm diameter, 82 ml volume, PIERCE)with 100 ml of 10 mM sodium phosphate buffer (wash buffer).

12. Load sample and follow with 60 ml of wash buffer.

13. Elute with 0.5M NaCl in wash buffer and collect 2 ml fractions.

14. Test fractions for absorbance at 280 nm using the elution buffer asa blank and cytotoxicity on Vero cells and pool appropriate fractions.

15. Clean column with 25 ml each of 8M Urea in wash buffer and 1M NaClin wash buffer.

16. Reequilibrate column with 100 ml of wash buffer and add azide to thetop of the column for storage.

17. Dialyse peak fractions against 4 L 0.01M Tris-CL (pH 7.0, SIGMA).

18. Lyophilize sample and resuspend in 1-2 ml dH₂O (OPTIONAL).

19. Do protein assay (BCA Protein assay reagent, PIERCE) and runeSDS-PAGE gel (Schagger, H. and von Jagow, G.; Analytical Biochem 166,368-379 (1987): 10% T table 2; first line table 3) to check purity.

Solutions:

HA column potassium phosphate buffer (0.5M stock) 17.42 g K₂HPO₄ up to300 ml with dH2O 6.8 g KH₂PO₄ pH 7.2 with KOH CF column Histidine buffer(0.025M) 2.0 g/500 ml H₂O pH 6.2 with HCI CB column Sodium phosphatebuffer (Wash buffer-WB) 0.71 g/500 ml Na₂HPO₄ pH 7.2 with HAc degasElution buffer (0.5M) Cleaning Buffers 2.922 g NaCI/100 ml WB 12.01 gUrea/25 ml WB 1.46 g NaCI/25 ml WB 0.01 M Tris 4.84 g Trizma Base 4 LddH₂O pH to 7.2 with HCl

Purification of VT2c from E32511

Pellet Preparation:

1. Prepare 3×2 L penassay broth (Antibiotic Media 3, DIFCO; pH 7.0) in3×5 L jugs and autoclave at 121° C. for 20 minutes. Allow broth to coolto room temperature before use.

2. Seed minimum 3×2 ml of penassay broth with E32511 and incubateovernight at 37° C.

3. Add 0.2 μg/ml Mitomycin C (1 ml of 0.4 mg/ml) (add 5 ml of ddH₂O tothe vial) to each of the 5 L jugs (from step 1). Seed each jug with 2 mlof seed (step 2) and incubate for 6 hrs at 37° C. with shaking ofapproximately 120 rpm. It is very important to stagger the incubation byabout 45 min/flask because the toxin begins to deteriorate after 6 hourexposure to Mitomycin C.

4. Spin down culture solution at 9,000× g for 15-20 min at 4° C. Discardsupernatant and store pellets at −20° C.

Preparation of Crude Toxin Extract:

1. Resuspend pellets in 150 ml of PBS (Phosphate buffered saline, OXOID;pH 7.3).

2. Add 0.3 mg/ml PMSF (phenylmethyl-sulfonyl fluoride, SIGMA) dissolvedin 0.5 ml acetone to pellet solution. Let acetone evaporate. Sonicate onice at highest output possible for 3 min or until an homogeneoussolution is obtained.

3. Add 0.1 mg/ml polymyxin B sulphate (Aerosporin, BURROUGHS WELLCOMEINC.; 500,000 units) to solution and incubate with gentle shaking at 37°C. for 1 hr.

4. Spin down cells at 9,000× g at 4° C. for 20 min (to remove all cellsand cell debris from solution).

5. Decant supernatant and store at 4° C. Resuspend pellet in 75 ml PBSand add 0.1 mg/ml polymyxin B.

6. Incubate with gentle shaking at 37° C. for 1 hr.

7. Spin down cell at 9,000× g at 4° C. for 20 min and pool supernatants(from step 5). Discard pellets.

The next few steps should preferably be done at 4° C.:

8. Add crystalline ammonium sulphate very slowly, with stirring topooled supernatants to 30% saturation.

9. Let stir for 20 min and then remove precipitate by centrifugation(10000 g for 10 min).

10. Add crystalline ammonium sulphate very slowly, with stirring topooled supernatants to 70% saturation.

11. Let stir for 20 min and then centrifuge at 10000 g for 10 min.

12. Resuspend pellet from step 11 in 15 ml of 0.01M Potassium phosphatebuffer.

13. Using 12-14,000 MW cutoff tubing (SPECTRAPOR) (now and in alldialysis steps), dialyse toxin solution against 4 L of 10 mM potassiumphosphate overnight, with stirring at 4° C.

Chromatography:

Hydroxylapatite (HA)

1. Equilibrate hydroxylapatite column (BSA binding capacity: 32 mg/g,approximately 113 ml volume; CALBIOCHEM (BEHRING DIAGNOSTICS)) with 2column volumes of 10 mM potassium phosphate.

2. Load sample and follow with 1 column volume 10 mM potassiumphosphate.

3. Add 2 column volumes of 100 mM-200 mM potassium phosphate and collect2 ml fractions. The fractions containing the toxin should be coloureddifferently from the other fractions.

4. Wash column with 1 column volume of 500 mM potassium phosphate andreequilibrate with 1 column volume of 10 mM K phosphate. Add azide tothe top of the column for storage.

Chromatofocussing (CF)

5. Pool peak fractions from HA column either by colour or bycytotoxicity test on Vero cells (10-fold dilutions).

6. Dialyse pooled fractions against 4L 0.025M imidazole-HCl pH 7.4(SIGMA) overnight. Also equilibrate the chromatofocussing column (PBE(polybuffer exchanger) 94, 1.5 cm diameter, 57 ml volume; PHARMACIA)overnight with the same buffer (300 ml).

7. Load sample and follow with 200 ml polybuffer-HCl pH 5.0 (25 mlpolybuffer 74 (PHARMACIA)+175 ml dH₂O−pH to 5.0 with HCl).

8. Collect 2 ml fractions and test the pH of each fraction. Once the pHhas dropped to 5.95, stop collecting fractions. Test the fractions forcytotoxicity on Vero cells (10-fold dilutions).

9. Pool peak fractions.

10. Clean column with 200 ml 1M NaCl. If really dirty follow with 100 ml1M HCl, but quickly equilibrate column with 0.025M imidazole.

Cibachron blue (CB)

11. Equilibrate cibachron blue (2 cm diameter, 82 ml volume, PIERCE)with 100 ml of 10 mM sodium phosphate buffer (wash buffer).

12. Load sample and follow with 60 ml of wash buffer.

13. Elute with 0.5M NaCl in wash buffer and collect 2 ml fractions.

14. Test fractions for absorbance at 280 nm using the elution buffer asa blank and cytotoxicity on Vero cells and pool appropriate fractions.

15. Clean column with 25 ml each of 8M Urea in wash buffer and 1M NaClin wash buffer.

16. Reequilibrate column with 100 ml of wash buffer and add azide to thetop of the column for storage.

17. Dialyse peak fractions against 4 L 0.01M Tris-CL (pH 7.0, SIGMA).

18. Lyophilize sample and resuspend in 1-2 ml dH₂O (OPTIONAL).

19. Do protein assay (BCA Protein assay reagent, PIERCE) and runSDS-PAGE gel (Schagger, H. and von Jagow, G.; Analytical Biochem 166,368-379 (1987): 10% T table 2; first line table 3) to check purity.

Solutions:

HA column potassium phosphate buffer (0.5M stock) 17.42 g K₂HPO₄ up to300 ml with dH2O 6.8 g KH₂PO₄ pH 7.2 with KOH CF column imidazole buffer(0.025M) 0.851 g/500 ml H₂O pH 7.4 with HCI CB column sodium phosphatebuffer (Wash buffer-WB) 0.71 g/500 ml Na₂HPO₄ pH 7.2 with HAc degasElution buffer Cleaning Buffers 2.922 g NaCI/100 ml WB 12.012 g Urea/25ml WB 1.461 g NaCI/25 ml WB 0.01 M Tris 4.84 g Trizma Base 4 L ddH₂O pHto 7.2 with HCl

Affinity purification verotoxins

500 μg globotriaosyl ceramide in 1 ml chloroform was mixed and driedwith 1 g of dried celite. The chloroform was evaporated and the celitesuspended in PBS and poured in a column. Crude polymyxin extract 20 ml(25 mg protein) the toxin producing E. coli was applied to the columnand incubated at room temp for 15 mins. The column was washed with PBSand purified verotoxin eluted with 10 ml 1M Tris pH 9.6. The eluate wasneutralized and dialysed. This method is applicable for purification ofall verotoxins. (Boulanger, J., Huesca, M., Arab, S and Lingwood, C. A.“Universal method for the facile production of glycolipid/lipid matricesfor the affinity purification of binding ligands” Anal Biochem 217: 1-6[1994])

Preparation of verotoxin 1 doses

VT1 was purified from the E. coli strain as previously described whichoverexpresses the cloned toxin genes. The purified toxin was free ofendotoxin contamination. The protein concentration of this batch ofverotoxin was determined and the toxin aliquoted and stored at −70° C.

To prepare VT1 doses for patients, VT1 was diluted into injection gradesterile saline containing 0.2% v/v of the patient's own serum. 210 ul ofsterile patient serum was added to 10 ml of sterile injection saline and93.9 ml of purified VT1 (6.7 g/ml) added to give a final toxinconcentration of 62.5 ng/ml or 12.5 ng per 0.2 ml. dose. The final toxinpreparation was sterile-filtered using a 0.2 mm syringe filter anddispensed in 2 ml aliquots into 10 ml vials. One working vial may bestored at 4° C. and the remaining vials frozen until needed.

FITC labelling of VT1: FITC was added directly to VT1 (in a 1:1, w/wratio) in 0.5M Na₂CO₃/NaHCO₃ conjugated buffer pH 9.5 and the mixturegently rotated for 1.2 hours at room temperature. Free FITC was removedby centricon.

Fluorescent Staining of Sections: Samples of surgically removed ovariantumours were embedded in OCT compound, flash frozen in liquid nitrogen,and stored at −70° C. until use. Five μm sections of frozen sample werethawed, allowed to dry and stained with FITC-labelled VT1 in PBS (0.5mg.ml) containing 0.1% BSA for 1 h at room temperature. Sections wereextensively washed with PBS and mounted with mounting medium containingDABCO. Sections were observed under a Polyvar fluorescent microscope.

Fluorescent Staining of Cells: Cells growing on coverslips were washedonce with PBS, fixed for 2 min at room temperature with 2% formalinrinsed with PBS twice and incubated with FITC-VT1 for 1 h at roomtemperature. The cells were washed 5 times with PBS, mounted with DABCOand observed under a Polyvar fluorescent microscope.

Quantification of VT1 antitumour activity: SKOV3 (drug sensitive humanovarian cell line), SKOVLC (SKOV3, resistant to Vincristine, and SKOVLB(SKOV3, resistant to Vinblastine) were each grown in α-MEM supplementedwith 10% fetal calf-serum and tested for their sensitivity to VTs. Equalnumbers of cells (approximately 1000 per/ml of media) were added to thewells of Linbro 98 well plate. 10-fold dilution of VTs were tested intriplicate and incubated for 48 h at 37° C. in a humidified atmospherecontaining 5% CO₂. Cells were then fixed with 2% Formalin, stained withCrystal Violet, and read with ELISA plate reader.

To quantify the anticancer activity of VT1, SKOV3, SKOVLC, and SKOVLB(human ovarian cell line) were incubated with 10-fold dilution of VT1for 48 h. SKOVLC & SKOVLB (drug resistant cell lines) are more sensitiveto VT1 antitumour activity than SKOV3.

Preparation of ¹³¹I-VT1B

This material may be made by the following procedure.

1. Dissolve 20 mg of iodogen in 2.0 ml of chloroform (10 mg/ml). Make a1:10 dilution by adding 0.25 ml of the 10 mg/ml solution to 2.25 mlchloroform (1 mg/ml).

2. Dispense 20 ul of this dilute solution into a clean, dry sterilizedglass tub. Add 500 ul of chloroform and evaporate to dryness under N₂.

3. Add 1.5 mg. in 0.66 ml of VT1B subunit to the test tube.

4. Add 5 MCi of ¹³¹I sodium iodide in 100 ul. Allow labelling to proceedfor 10 mins.

5. Wash a PD-10 column with 25 ml of Sodium Chloride Injection USP.

6. Dilute ¹³¹I-VT1B to 2.5 ml total volume with 1% HSA in SodiumChloride Injection USP. Load onto PD-10 column. Elute column with 3.5 ml1% HSA in saline.

7. Measure ¹³¹I activity of eluant and column to determine LE. Draw uppooled fractions into a syringe with spinal needle attached. Detachspinal needle and attach Millex GV filter.

8. Filter into a sterile 10 ml multidose vial. Note volume filtered andassay vial for ¹³¹I in dose calibrator. Calculate concentration.

9. Draw up 0.1 ml of ¹³¹I-VT1B and dispense 0.05 ml into each of two 5ml sterile multidose vials (one for sterility test and one for pyrogentest). Vials already contain 2 ml saline (=1:50 dilution).

10. Determine RCP by PC (Whatman No. 1) in 85% MeOH and by sizeexclusion HPLC.

11. Conduct sterility and pyrogen tests.

FIG. 1 relates to the neutralization of ACP cytotoxicity by anti-VT. KHTcell monolayers were incubated with 35 ng/ml ACP from E. coli HSC₁₀, or10 pg/ml VT1, VT2 or VT2c in the presence of monoclonal anti-VT1(PH1),monoclonal anti VT2 or polyclonal rabbit antiVT1 B subunit. The cellswere incubated for 72 hours at 37° C. and viable adherent cells weredetected by fixation and staining with crystal violet. Cytotoxity of VT1and ACP was completely neutralized in the presence of anti VT1 or antiVT1B subunit (anti-VT2 serum had no effect).

From measurement of the cytotoxic assay of ACP on vero cells (cells fromAfrica green monkey kidney that are very sensitive to verotoxin),relative to a pure VT1 standard, it was estimated that the ACPpreparation contained 0.05% VT1. This concentration of purified VT1 wasas effective as ACP in inhibiting the growth of various tumour celllines in vitro (FIG. 2). Thus, VT1 mimics the anti-neoplastic effect ofACP in vitro. VT1 was tested for the ability to inhibit the metastasesof KHT fibrosarcoma cells in the mouse model as had been previouslyreported for ACP. The equivalent dose of VT1 was as effective as ACP,reducing the number of lung metastases to background levels, following aprimary subcutaneous tumour inoculum (Table 1).

TABLE 1 Response of KHT cells, growing as lung modules, to treatmentwith VT-1 or ACP. # OF # OF LUNG WT LOSS GP TREATMENT MICE NODULES/MOUSEMEAN /GAIN* EXPT 1 1 Control 9 34, 24, 39, 47, 28 32.6 +5% , 32, 26, 29,34 2 ACP-0.25 ug/mouse 4 12, 31, 25, 15 20.8 0 3 ACP-1.0 ug/mouse 6 1,2, 2, 5, 1 2.2 0** (1 death) 4 ACP-4 ug/mouse 5 0, 0, 0, 0, 0 0 −13%  5VT-1 0.009 ug/mouse 5 29, 41, 34, 29, 21 30.8 +5% 6 VT-1 0.036 ug/mouse5 7, 16, 29, 16, 6 14.8 +5% 7 VT-1 0.144 ug/mouse 5 1, 4, 2, 3, 1 2.2+5% EXPT 2 1 Control 4 15, 12, 8, 12 11.7 <5% 5 2 ACP-2 ug/mouse 5 0, 1,0, 0, 0 0.2 <5% 3 VT-1 0.1 ug/mouse 4 0, 0 0 <5% (2 deaths) 4 VT-1B-0.2ug/mouse 5 13, 14, 9, 7, 19 12.4 <5% 5 VT-1B-10 ug/mouse 5 8, 3, 9, 116.8 <5% Mice were treated with VT-1 or ACP(1-p) I day after cellinjection (1000 KHT cells/mouse i-v). Lung nodules counted @ 20 daysafter cell injection. *Mean change in gp wi-miax during 10 days (Expl 1)or 4 days (Expt 2) after VT-1 or ACP injection. Max wt loss @ days 7-8.**Death occurred @ days 2-3 after ACP injection ***Deaths occurred @days 7-8

Purified VT1 was found to mimic the anti-metastatic effect of ACP on thegrowth of this tumour from a primary subcutaneous site. Lung metastasiswas completely inhibited. Moreover, prior immunization of mice with thepurified B-subunit of verotoxin completely prevented any protectiveeffect of ACP when the animals were subsequently treated with the tumourand ACP (Table 2).

TABLE 2 Response of KHT lung nodules, growing to immunized mice, totreatment with VT1 or ACP. # OF LUNG IMMUNI- # OF NODULES/ WT LOSS/ GPZATION* TREATMENT MICE MOUSE MEAN GAIN* 1 None None 6 34, 47, 53, 48.5<5% 62, 43, 52 2 None VT-1 -0, 2 5 5 deaths ug/mouse (dy 6-8)** 3 NoneACP-2.0 5 0, 1, 2, 0, 0 0.6 −8% ug/mouse 4 VT-1B + FA None 5 43, 40, 47,39.2 −6% 43, 23 5 VT-1B + FA VT-1 −0, 2 6 26, 44, 49, 36.7 <5% ug/mouse21, 43, 37 6 VT-1B + FA ACP-2.0 6 50, 38, 33, 43.3 <5% ug/mouse 41, 48,50 7 FA only None 5 44, 60, 19 37.6 <5% 25, 40 8 FA only VT-1 −0, 2 5 5deaths ug/mouse (dy 6-8)*** 9 FA only ACP −2.0 5 1, 1, 2, 1, 0 1 −6%ug/mouse Mice were treated with VT-1 or ACP(i-p) 1 day after cellinjection (100 KHT cells/mouse). Lung nodules counted @ 20 days aftercell injection (i-v). *Immunization was 2 injections of VT-1B (10ug/mouse +/− Freund's Adjuvant (FA) given (i-p) 4 weeks and 2 weeksbefore cell injection. **Mean change in gp wt - max during 13 days.Maximum weight loss @ day 7-8.

ACP was tested for glycolipid binding by TLC overlay using monoclonalanti-VT1 or anti-VT2c. Anti-VT1 shows extensive binding of a componentwithin the ACP preparation to globotriaosylceramide and galabiosylceramide (FIG. 3). This binding specificity is identical to thatreported for purified VT1(8). No binding component reactive withanti-VT2 was detected. In FIG. 3 anti VT antibodies were used to detectbinding to the immobolized glycolipids. Arrows indicate position ofstandard (from the top) galabiosyl ceramide, globotriaosyl ceramide andglobotetraosyl ceramide. Panel 1-detection using anti VT1, panel2-detection using anti VT2c.

VT1 demonstrated in vitro activity against a variety of ovariancarcinoma cell lines. A large number of primary human ovarian tumourbiopsies were screened for the expression of Gb₃ via TLC overlay usingpurified VT1. It was found that Gb₃ was barely detectable in normalovary tissue, whereas in all cases a significant increase in expressionof Gb₃ was observed in the ovarian carcinoma. Similarly, elevated levelsof Gb₃ were found in acites tumour and in tumours that had metastized tothe omentum, (FIG. 4) which defines lane 1, ovarian omentum metastasis;lane 2: tumour biopsy; lane 3, tumour biopsy; lanes 3-6, normal ovary;lane 7, human kidney Gb₃ standard. Surprisingly, we have found thatmulti-drug resistant variants of ovarian tumour cell lines wereconsiderably more sensitive to VT1 cytotoxicity than the drug sensitiveparental cell line (FIGS. 2, 5 and 6). Similar effects had been observedfor ACP. FIG. 2 shows human ovarian tumour cell lines sensitive to ACPtested for VT sensitivity. Human ovarian and breast tumour derived celllines were tested for VT1 sensitivity wherein ovarian 1, 2, 3, 4 and 5are denoted , +, ×, ▪ and o respectively, and breast-SKBR3Δ, 468♦, 453,231▴. The cell lines 1-ovarian, 453 and SKBR3, previously shown to beresistant to ACP, were also resistant to up to 20 ng/ml VT1.

The 1, 2, 3 and 4 cells were from ovarian cancer patients; the 453 cellswere from a breast cancer patient; 231 and SKBR3 are breastadenocarcinoma cell lines, and 5, SKOV3 and SKOVLB are adenomacarcinousovarian cancer cell lines. The lines 1, 453 and SKBR3, resistant to ACP,were co-resistant to VT1. FIG. 5 shows VT sensitive and resistant celllines tested for the presence of Gb₃ by VT binding in tlc overlay.Glycolipid from an equal number of cells were extracted and separated bytlc prior to toxin binding. In FIG. 5, lane 1:SKBR3, lane 2:468, lane3:231, lane 4:453, lane 5 Gb₃ standard, lane 6:SKOV3, lane 7:SKOVLB.Cell lines SKBR3, 468, 231 and 453 are derived from breast tumours. Only231 is sensitive to VT1. SKOVLB is a multiple drug resistant ovariantumour cell line derived from SKOV3.

Ovarian tumour cells were highly sensitive to VT (FIG. 3) and containedelevated levels of the VT receptor, Gb₃ (FIG. 4). Breast cancer cellswere for the most part, toxin resistant (FIG. 3) and receptor negative(FIG. 5). Low levels of Gb₃ were detected in normal ovarian tissue butthese were markedly elevated for the ovarian tumour tissue samples.

The specific elevation of Gb₃ in ovarian tumours as opposed to normalovary tissue provides the feasibility of using the toxin in themanagement of this malignancy. Ovarian tumours are often refractory tochemotherapy and prognosis is poor. Indeed, preliminary phase 1 clinicaltrials using a ACP injected directly into skin malignancies (Mycosisfungoides) have proven successful without adverse systemic effects.

With reference now to FIG. 6, human derived ovarian tumour cell lineswere tested for VT1, VT2, and VT2c sensitivity. The cells were grown toconfluence in 48-well plates, then incubated for 48 hrs. in the presenceof increasing doses of VTs. SKOVLB, the multiple drug resistant variantof SKOV3 ovarian line, showed the most sensitivity to VT's with SKOVLCbeing the next most sensitive to the VT's.

We have found that both drug resistant cells are approximately 500 to1000 times more sensitive to verotoxin cytotoxicity than the parentalSKOV3 cell line.

FIG. 7 shows the effect after 48 hrs. of treatment of the brain tumourSF-539 cell line derived from a recurrent, right temporoparictalglioblastoma multiform with VT1, VT2, and VT2c. This cell line, asothers, was highly sensitive to VT's.

FIG. 8 provides the results from imaging a nude mouse with ¹³¹I-VT1B(CPM distribution in different organs). VT1B-¹³¹I cpm distribution innude mouse with implanted ovarian tumour showed that a considerableamount of radiolabled VT1B had been concentrated in the ovarian tumour.only a trace amount of VT1B was located in the brain where the potentialVT1 side effect was considered. Since the lung in human adult is not thesite of concern for VT1 toxicity this does not present a problem fortreatment of human adult with ovarian tumour. In addition the CPM inkidney includes the excreted radiolabelled VT1 B subunit. Accordingly,based on this test, imaging with labelled VT1 B subunit can be a veryuseful method for screening the susceptible patient to VT1 cytotoxicity.

FIG. 9 shows the sensitivity of a variety of human astrocyta cell linesto VT1. All these cells contain Gb₃ but show variable sensitivity to VT1induced cytotoxicity. This suggests that certain astrocytomas will besusceptible to verotoxin wheres others may not. This is important sinceastrocytomas are very refractory to treatment at the present time andcell sensitivity in vitro to concentrations as low as 5 ng per/ml israre.

While the invention has been described in detail and with reference tospecific embodiments thereof, it will be apparent to one skilled in theart that various changes and modifications can be made therein withoutdeparting from the spirit and scope of the invention.

We claim:
 1. A method of treating mammalian neoplasia comprisingtreating a mammal in need of such treatment with a non-lethal,anti-neoplasia effective amount of a verotoxin, wherein said mammal is ahuman, and wherein said neoplasia comprises Gb3-containing cells.
 2. Amethod as defined in claim 1 wherein said verotoxin is verotoxin
 1. 3.The method of claim 1, wherein said method of treating comprisesadministering said verotoxin and a pharmaceutically acceptable diluent,adjuvant or carrier.
 4. The method of claim 3, wherein said verotoxin isadministered at a concentration of about 62.5 ng/ml.
 5. The method ofclaim 3, wherein said verotoxin is administered at a concentration ofabout 12.5 ng/0.2 ml.
 6. A method of treating mammalian neoplasiacomprising treating a mammal in need of such treatment with anon-lethal, anti-neoplasia effective amount of a verotoxin, wherein saidverotoxin is verotoxin 2, and wherein said neoplasia comprisesGb₃-containing cells.
 7. A method of treating mammalian neoplasiacomprising treating a mammal in need of such treatment with anon-lethal, anti-neoplasia effective amount of a verotoxin, wherein saidverotoxin is verotoxin 2c, and wherein said neoplasia comprisesGb₃-containing cells.
 8. The method of claims 6 or 7, wherein saidmammal is a human.
 9. The method of claims 1, 6, or 7, wherein saidneoplasia is multi-drug resistant.